1. Technical Field
The present invention is concerned with the ex vivo, cell-mediated, delivery of pharmaceutically active material to a subject. The pharmaceutically active material is initially prepared as a microparticulate, which is engulfed or adsorbed by cells. The cells are then administered to a subject.
2. Background Art
The ex-vivo delivery of pharmaceutically active compounds has been successfully accomplished in several therapeutic areas, particularly in gene delivery. For ex-vivo delivery, cells are removed from the patient, modified outside the body, and reimplanted. In-vivo delivery consists of administering the pharmaceutical agent directly into the body, including intravenous, intramuscular, subcutaneous, intradermal, intra-articular, intrathecal, epidural, intracerebral, bucchal, rectal, topical, transdermal, oral, intranasal, pulmonary, and intraperitoneal administration. In-vivo delivery of genetic material is, at first glance simpler, but raises significant safety concerns. Ex-vivo delivery is more complicated but permits safety testing before the genetically modified cells are administered. Candidate genetic material includes encoding for secreted factors that could have broad applications ranging from treatment of inherited single-gene deficiencies to acquired disorders of the vasculature or cancer. Myoblasts have been recently transfected via retroviral vectors (Ozawa C R, Springer M L, Blau H M, A novel means of drug delivery: myoblast-mediated gene therapy and regulatable retroviral vectors. Annu Rev Pharmacol Toxicol. 2000; volume 40, pages 295-317).
In the area of pharmaceutical delivery of small molecules, Bender et al. disclose the treatment of HIV-infected monocytes/macrophages with polyhexylcyanoacrylate nanoparticles loaded with either the nucleoside analog zalcitabine (2′,3′-dideoxycytidine), or saquinavir, a protease inhibitor (Bender et al., Efficiency of Nanoparticles as a Carrier System for Antiviral Agents in Human Immunodeficiency Virus-Infected Human Monocytes/Macrophases In Vitro, Antimicrobial Agents and Chemotherapy, June 1996, volume 40(6), p. 1467-1471). The polyhexylcyanoacrylate nanoparticles were prepared by emulsion polymerization and tested in-vitro for antiviral activity in primary human monocytes/macrophages. An aqueous solution of saquinavir showed little antiviral activity in HIV-infected macrophages, whereas the nanoparticulate formulation demonstrated significant antiviral activity at one-tenth the solution concentration. At a concentration of 100 nM, saquinavir in solution was completely inactive in chronically HIV-infected macrophages, but when bound to nanoparticles it caused a 35% decrease in viral antigen production. In this study, the drug was entrained in a polymer (polyhexylcyanoacrylate) matrix. Preparation of pure, solid drug nanoparticles for delivery to macrophages was not disclosed. Particles were only delivered to macrophages in-vitro and did not contemplate drug delivery by administering nanoparticle-treated cells that are capable of transporting the drug.
U.S. Pat. No. 4,973,465 (Baurain et al.) and U.S. Pat. No. 5,100,591 (Leclef et al.) discloses lipid microparticles of nystatin, amphoterin B and other anti-fungal compounds, potentially having enhanced targeting for macrophages.
The present invention overcomes the disadvantages of the prior art by providing pharmaceutical compositions comprising solid pharmaceutical agents in an ex-vivo drug delivery method. The advantage to this approach over the use of an adjunct (i.e., direct patient dosing formulation) matrix, is that high drug loading is achieved and a high burst of drug may be delivered. As part of this invention we disclose the ex-vivo application of solid submicron particles that are mostly free of a carrier matrix and consisting only of solid drug with stabilizing surface-active ingredients. The solid particulate is contacted with cells capable of engulfing the particles or in which the particles are capable of attachment to the outer surfaces of the cells. Several therapeutic areas are appropriate for this ex-vivo technology: treatment of bacterial, viral, and fungal infections, neoplasms, lysosomal storage disorders, autoimmune disorders, and metabolic disorders. Gene delivery and delivery of antisense oligonucleotides are also disclosed.
Ex-vivo delivery of solid nanoparticles, particularly particles of essentially pure active agent, can provide many advantages in several therapeutic areas. For example, in gene therapy, vectors are typically used that can either be viral or nonviral. To their advantage, viruses offer strong specificity and high transfection efficiency via the virus's natural mechanism for delivering DNA into the cell. Having the gene expressed in-vivo can also induce unintended, fatal consequences, particularly if undesired expression of the viral genetic material occurs. Nonviral approaches, on the other hand, though less toxic, are relatively inefficient and nonspecific. However, the non-viral vectors rely on carrier vesicles for the nucleic acid. U.S. Patent Publication No. US 2003/0092069 attempts to remedy the non-specific delivery of genes, in vivo, in disclosing the site specific delivery of genes via a hollow nanoparticle. More specifically, the '069 publication discloses the use of a biorecognition molecule (L protein of hepatitis B virus) for the delivery of a protein to a hepatocyte.
Pharmaceutical agents may be delivered to phagocytizing cells in ex-vivo culture by adding a solution. However, most of the drug may not sufficiently concentrate in cells when influx relies on molecular diffusion of drug solute across membranes. This inefficient utilization of drug may require extracellular perfusion of higher drug concentrations that can produce cytotoxic effects in culture. More efficient delivery of the drug may be realized by leveraging the ability of phagocytes (e.g., macrophages, monocytes, reticulocytes, eosinophils, basophils, neutrophils, and dendritic cells, among others) to engulf particles.
The present invention overcomes the foregoing limitations by delivering a substantially carrier-free, pharmaceutical agent microparticulate, by cells that are capable of engulfing the microparticulate, or by adsorbing the pharmaceutical agent microparticulate on the cell surface, and which upon delivery of the loaded cells to the patient is capable of reaching the target tissues.